Ψ-Footprinting approach for the identification of protein synthesis inhibitor producers

Abstract Today, one of the biggest challenges in antibiotic research is a targeted prioritization of natural compound producer strains and an efficient dereplication process to avoid undesired rediscovery of already known substances. Thereby, genome sequence-driven mining strategies are often superior to wet-lab experiments because they are generally faster and less resource-intensive. In the current study, we report on the development of a novel in silico screening approach to evaluate the genetic potential of bacterial strains to produce protein synthesis inhibitors (PSI), which was termed the protein synthesis inhibitor ('psi’) target gene footprinting approach = Ψ-footprinting. The strategy is based on the occurrence of protein synthesis associated self-resistance genes in genome sequences of natural compound producers. The screening approach was applied to 406 genome sequences of actinomycetes strains from the DSMZ strain collection, resulting in the prioritization of 15 potential PSI producer strains. For twelve of them, extract samples showed protein synthesis inhibitory properties in in vitro transcription/translation assays. For four strains, namely Saccharopolyspora flava DSM 44771, Micromonospora aurantiaca DSM 43813, Nocardioides albertanoniae DSM 25218, and Geodermatophilus nigrescens DSM 45408, the protein synthesis inhibitory substance amicoumacin was identified by HPLC-MS analysis, which proved the functionality of the in silico screening approach.

: Composition of the production media used for cultivation of the antibiotic producers. All quantities refer to 1 l.

Medium
Composition Amount

Supplementary Figure S3:
Chemical structure of berninamycin derivatives. Berninamycin A -hydroxy group at Val7; berninamycin B -missing hydroxy group at Val7; berninamycin C -one missing dehydroalanine residue at the N-terminus, berninamycin D -two missing dehydroalanine residues at the N-terminus; berninamycin E -in place of the N-terminal dehydroalanine is an alanine residue (2,4) Purple ellipses indicate the position of the altered berninamycin residues. Dehydroalanine (Dha), alanine (Ala), valine (Val). Figure S3: Additional information: Berninamycin BGC comparisons provide information on the production of compound derivatives:

Supplementary
Tü 2108 was found to produce berninamycin C exclusively (5) ( Figure S2). S. bernensis produces berninamycin A-D, with berninamycin A as the major compound (6), whereas heterologous expression of the berA-J gene cluster from S. bernensis in Streptomyces lividans resulted in a stable berninamycin A production, while berninamycin B and D were only produced in tiny amounts and berninamycin C was not produced at all (6). S. atroolivaceus produces berninamycin A and E, with berninamycin A as the major compound (4,7). Differences in berninamycin derivative production might be related to the berA core-prepeptide coding sequence. Malcolmson et al., 2013 showed that mutations in the BerA core-prepeptide sequence result in the production of berninamycin analogs, e.g. the introduction of a T3A mutation in the berA gene sequence resulted in the production of different compounds in S. lividans, including a substance with a Cterminal amide, an unmodified Val7 residue, and an N-terminal alanine residue (6). Thus, berA mutations cause structural changes in the BerA protein, which are responsible for the formation of the various different berninamycin derivatives (2, 4) ( Figure S3). Since the similarity between BerA from S. bernensis and ORF 01237 from Tü 2108 is rather low (only 65%), the exclusive berninamycin C production of Tü 2108 is likely to be the result of the specific core prepeptide sequence encoded by ORF 01237. Thus, the results presented in this study show that Tü 2108 is a new producer strain of berninamycin and provide an explanation for the high berninamycin C production rate of Tü 2108. Table S3: Sum of the 790 detected hit genes from 35 analyzed genome sequences of known PSI producers and their assignment to the following criteria for selfresistance genes: phylogenetic incongruence of core gene, gene duplication, gene localization in a BGC. Shown are the gene numbers and the percentage for each criterium. Some of the genes fulfill several criteria; therefore, the total numbers and percentage of the genes exceeds 790 and 100%, respectively.  Figure S4: In vitro transcription/translation assay performed with culture extracts in MS (A) and R5 (B) media from 4, 7, and 10 days. PSI antibiotics, tet15 and apra50 = positive control (orange), medium extracts, extracts of M1146 = negative control (green), and extracts of DSM cultures (blue). Results are shown for samples, which did not lead to an inhibition of the ivTT assay. Measurements have been performed in triplicate using the same preparation of S12 extract.

No. of genes Percentages of genes
Supplementary Figure S5: In vitro transcription/translation assay performed with culture extracts in NL800 (A), MS (B), and R5 (C) media from days 4, 7, and 10. Only strains are shown, which were able to grow in the respective media. PSI antibiotics, tet15 and apra50 = positive control (orange), medium extracts, extracts of M1146 = negative control (green), and extracts of DSM cultures (blue). Measurements have been performed in triplicate using the same preparation of S12 extract.

Supplementary Figure S5 -Additional information: Description of additional in vitro transcription/translation (ivTT) assay results:
IvTT assays with extract samples of strain DSM 44944 incubated for 10 days in NL800 medium resulted in inhibition of GFP expression with values of 29% maximal fluorescence ( Figure 6, Supplementary Figure S5). Extract samples of strain DSM 45888, DSM 45258, and DSM 43821 grown for 7 days in NL800 showed similar values (28%, 26%, and 24%, respectively) ( Figure 6, Supplementary Figure S5). Inhibition of the ivTT assay suggests that DSM 44944, DSM 45888, DSM 45258, and DSM 43821 produce a PSI when grown in NL800, whereby the time point for best production varies. Measurements have been performed in triplicate using the same preparation of S12 extract.
Supplementary Figure S8: In vitro transcription/translation assay performed with R5 culture extract of DSM 45408 from day 10. Displayed are the fractions generated by semipreparative HPLC (blue). PSI antibiotic apra50 = positive control (orange). Fraction having the greatest inhibition is shown in red Measurements have been performed in triplicate using the same preparation of S12 extract.
Supplementary Figure S9: In vitro transcription/translation assay performed with R5 culture extract of DSM 25218 from day 7. Displayed are the fractions generated by semipreparative HPLC (blue). PSI antibiotic apra50 = positive control (orange). Fraction having the greatest inhibition is shown in red Measurements have been performed in triplicate using the same preparation of S12 extract.
Supplementary Figure S10: In vitro transcription/translation assay performed with R5 culture extract of DSM 44771 from day 4. Displayed are the fractions generated by semipreparative HPLC (blue). PSI antibiotic apra50 = positive control (orange). Fraction having the greatest inhibition is shown in red Measurements have been performed in triplicate using the same preparation of S12 extract.

HRMS analysis
High resolution mass spectrometry was done with a Bruker Elute UHPLC 1300 coupled with a QTOF (Impact II, Bruker). 2 µl extract were injected on a Kinetex 100 C18 column (50 x 2.1 mm, 1.7 µm, Phenomenex) and eluted by a linear gradient from 90 % solvent A (water with 0.1 % formic acid) to 100 % solvent B (acetonitrile with 0.1 % formic acid) over 10 minutes with a flow rate of 0.5 ml/min. QTOF parameters were as follows: capillary voltage 4500 V, nebulizer 2 bar, drying gas 8 l/min, dry heater 220 °C. Data evaluation was conducted with Bruker Compass DataAnalysis 5.2. Figure S11:HRMS data of sample F8 from DSM 45408. Positive extracted ion chromatograms (EICs) and MS 1 of amicoumacin A (above) and B (below). Expected mass for amicoumacin A (C20H30N3O7) is 424.2078 and for amicoumacin B (C20H29N2O8) 425.1918, error rates are -0.1 ppm. Figure S12: Whole-genome sequence-based phylogenetic tree generated with the TYGS web server for the amicoumacin B producer strains DSM 44771, DSM 43813, DSM 25218, DSM 45408, and closely related type strains. The known amicoumacin producers Bacillus pumulus and Nocardia jinanensis were added manually to the reference strains. All amicoumacin producer strains are marked in bold. Tree inferred with FastME from GBDP distances calculated from genome sequences. The branch lengths are scaled in terms of GBDP distance formula d5. The numbers above branches are GBDP pseudo-bootstrap support values >62% from 100 replications, with an average branch support of 85.3%.